JPH11504808A - Measurement of glycated protein - Google Patents

Abstract

PCT No. PCT/EP96/01912 Sec. 371 Date Nov. 5, 1997 Sec. 102(e) Date Nov. 5, 1997 PCT Filed May 3, 1996 PCT Pub. No. WO96/34977 PCT Pub. Date Nov. 7, 1996The amount of glycated proteins in a sample can be quantified by reacting the sample with first a reagent which is a combination of a protease and a peroxidase and second with a ketoamine oxidase. A kit which contains the combined peroxidase/protease enzyme reagent and also the ketoamine oxidase is also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION                           Measurement of glycated protein   The present invention relates to the measurement of glycated proteins, and more particularly, to peroxidase. Measurement of glycated proteins, including the inclusion of proteinase in the same reagent as the enzyme It relates to a standard method and a kit for the measurement method.   Horseradish peroxidase is a redox enzyme (donor: hydrogen peroxide Reductase; EC 1.11.1.7). This peroxidase is Is widely used as an indicator enzyme [eg, Essays in Biochem istry, Vol. 28, pp. 129-146 (1994)]. It is a member of the oxidase family. A particularly useful feature of this enzyme is its carrier (e.g., (E.g., antibodies and other enzymes), high activity on a wide range of substrates Degree and good thermal stability. This enzyme has a single port containing 308 amino acids. It has a relative molecular mass of 44,000 and a hemin Combined with the group, it shows brown coloration. This enzyme has four disulfide bridges and two It has lucium ions, and its removal reduces stability.   This enzyme catalyzes the transfer of hydrogen from a hydrogen donor to a hydrogen acceptor. Hydrogen acceptor May be methyl peroxide or ethyl peroxide, but usually hydrogen peroxide is used. Can be. Hydrogen peroxide is reduced by the formula:           H_TwoO_Two  + AH_Two  → 2H_TwoO + A   A wide range of hydrogen donors are used, such as phenols, aminophenols, Indophenols, diamines, leuco dyes and the like can be mentioned. This kind of Products generated during the oxidation process to remove hydrogen from the compound visually detected? Or by a spectrophotometer. Fluorescence intensity as another detection method Method, emission photometry and electrochemical method.   Enzymes are physically linked to other proteins, such as antibodies and their fragments. This allows the analyte to be measured using the specific binding properties of the antibody, Alternatively, the location of the antigen may be histologically identified. This enzyme is called oxidase enzyme The oxidase substrate may be quantified by chemical coupling. Specific There are many analytes that can be measured using oxidase, but some It is contained in biological fluids, the analysis of which is clinically useful. Oki Phenols and amino acids as chromogens linked to sidase-peroxidase The use of ntipyrene has been known for a long time [see, for example, Ann. Clin. Bio chem. 6, Vol. 24, pp. 24-27 (1969)]. Relatively recently N-ethyl-N- (2-hydroxy-3-sulfopropyl)-in place of phenol m-toluidine (TOOS) etc. are more sensitive and color over a wide pH range [Eg Chem. Pharm. Bull. , Vol. 30, 249 2 to 2497 (1982)].   One such analyte is a glycated protein or fructosamine. This minute Deposits form glucose or other sugars that form condensation products with free amino groups of the protein. Which are the products of non-enzymatic reactions that occur [see, for example, Clin. Chem. , Vol. 33, No. 21 53 to 2163 (1987)]. The major glycated protein in blood is Albumin (in this compound, the exposed lysine residue results in a free amino group ) And hemoglobin (in this compound, the N-terminal valine amino acid is Also reacts to the course). In diabetic patients, protein components in blood The concentration of glucose varies within fairly narrow limits, while the concentration of glucose Change. Many pathological changes in diabetics are caused by long-term protein Exposure results from an increase in glucose concentration. Therefore, Evaluation of the average glucose exposure time over the life of the protein Measurement of protein is clinically useful.   Several methods have been used to measure total glycated protein. Current Is a furosine procedure [see, for example, J. et al. Clin. Chem. Clin. Biochem. 19, pp. 81-87 (1981)]. . In this method, the protein was digested with 6 molar hydrochloric acid at 95-100 ° C for 18 hours. Process is included. Furosine produces glycated lysine under these treatment conditions. Success And may be measured by HPLC. Use this method as a convention Is too complicated and time consuming. The thiobarbituric acid method has a short acid digestion time So (2-5 hours) is a somewhat simpler method. 5-Hydride obtained by this acid digestion Roxymethylfurfural reacts with thiobarbituric acid at 443 nm. This gives a derivative exhibiting a large absorbance. Another method is phenylboronate affinity This is a method using chromatography. Under alkaline conditions, phenylboro The nate forms a complex with the cis-diol group of the sugar. However, strict temperature control Even if the method is performed closely and glucose is removed in advance, the accuracy of this method Is low.   The simplest and most widely used method for measuring serum glycated proteins Is the nitroblue tetrazolium of fructosamine in an alkaline solution (N BT) [for example, Clin. Chem. Acta, Vol. 127 87-95 (1982)]. The big advantage of this method is that automation is It's easy. This method reduces interference from protein concentration, lipids and uric acid [See, for example, Clin. Chem. 37, 552 556 (1991)]. However, ordinary diabetics or enough Only about half of the reductive activity measured in diabetic patients Protein [see, for example, Clin. Chem. 34, pp. 320-32 3 (1988)].   To avoid the problem of poor specificity seen in the case of the NBT method, [Eg European Patent Application EP-A-526 150] See detailed book]. In this two-part reagent system, a protein that degrades serum proteins Use of ketoamine oxidase, which acts on saccharified fragments I do. This oxidase terminates when the amount of color development is proportional to the amount of glycated protein in the sample. Binds to peroxidase and chromogenic system in point determination. Enzymes from different sources A similar method has been proposed using [eg European Patent Application EP-A- No. 576 838]. However, these methods are suitable for fresh samples. When used, are susceptible to significant interference between ascorbate and bilirubin, and Since the second reagent contains peroxidase, blank correction of the reagent is required .   Fructosyl obtained by culture of Fusarium or Gibberella strains A method utilizing an amino acid oxidase has also been proposed [European Patent Application E P-A-678 576].   Proteinase in the first reagent has high activity on glycated proteins and ketoamine The ability to release the substrate for oxidase must be demonstrated. Rapid release of substrate In order to achieve this, it is advantageous to exhibit the ability to cleave different peptide bonds. Non-specific Some proteinases (eg, proteinase and proteinase K) and And other proteinases with different specificities from multiple admission sources They are known and may be used alone or in combination.   Due to the demand for high degradability of blood proteins and non-specific proteinases, It is almost impossible to retain the enzyme activity as a proteinase in the same solution. Not. Therefore, peroxidase must be contained in the second reagent of the two-part reagent. Therefore, the peroxidase exposure to the proteinase is caused by the second reagent. A relatively short time in the cuvette after the addition. The absorbance of the cuvette is Measured immediately before addition, and with the ketoamine oxidase / peroxidase system It is measured after coloring. Change in absorbance in these two measurements on the sample Is caused not only by the content of glycated protein in the sample but also by the absorbance of the second reagent itself I do. Therefore, a blank sample must be analyzed and corrected.   In order to be widely accepted as a clinical method, it is necessary to use a substrate other than the desired analyte. It is important to minimize interference due to this. Oxidase-peroxidase system Are two compounds that are known to interfere with bilirubin and bilirubin and ascorbate [For example, Ann. Clin. Biochem. 21, volume 398-404 ( 1984)]. The concentration of glycated protein in normal serum is about 0.1 mmol / L . Excess (3 ml / L) analyte (eg, glucose or choles) Interference with relatively low concentrations of analyte compared to (terol) is more severe. Large amounts of Bi Oral administration of Tamine C causes significant interference even in cholesterol assays [For example, Clin. Chem. 38, 2160 (1992)].   Other attempts have been made to reduce bilirubin interference. like this Some attempts have included reducing the pH during the reaction to 6.1 [see, for example, Clin. Chem. 27, pages 375-379 (1981)]. The necessary enzymes are Since higher pH is required, this method is used as an enzymatic assay for glycated proteins. Is not suitable. Alternatively, peroxidation that oxidizes peroxidase and bilirubin A method of pretreating a sample using hydrogen is also known [for example, Clin. Chem. , 38, pages 2411 to 2413 (1992)]. However, this The method uses an extra reagent, so that the subsequent oxidase reaction may interfere. I'm sorry. Bilirubin oxidase has been used to remove bilirubin [eg, Clin. Chem. 30, Volume 1389-1392 (1984)]. Only The bilirubin oxidase in the first reagent of the glycated protein assay Extra reagents cannot be maintained in the presence of proteinase. Required.   Interference effect of bilirubin up to 170 μmol / L in uric acid assay Potassium ferrocyanide has been used to remove [Clin. C hem. 26, 227-231 (1980)]. However, this In such cases, the effect of removing the interference is large, but potassium ferrocyanide is used in a two-part reagent system. The stability of the reagent is deteriorated because it is blended with the first reagent [for example, Eur. J. Clin . Chem. Clin. Biochem. 31, Volume 861, pp. 868 (1993). reference]. As described below, according to the present invention, glycated protein The addition of potassium ferrocyanide to the first reagent of the two-part reagent It is possible to remove the interference effect of bilirubin up to 00 μmol / L. The reagent was found to be stable at 4 ° C. for several weeks.   Protects the oxidase / peroxidase system from ascorbate interference Several methods are known for doing so. The most commonly used means are Scorbate oxidase [see, for example, Clin. Chem. , Volume 26, 2 27 to 231 (1980)]. However, this enzyme is When added to a reagent containing a protease, it rapidly decomposes, which is not suitable. Activated carbon The pretreatment method used [for example, Clin. Chem. 35, pp. 2330-233 3 (1989)] is inconvenient.   A color-forming substance equal to or greater than the redox potential of ascorbate Using metals having a redox potential lower than the redox potential of the substrate It is also known to eliminate interference effects due to acid salts. This kind of metal, including copper, And metals of groups VIII, IB, II-B and IV-A of the periodic table. See, for example, U.S. Pat. No. 3,411,887]. Redox electricity of metal ions It is important that the position is lower than the redox potential of the chromogenic substance, The ions themselves develop color in the absence of the analyte of interest. Similar oxidase / pe The effect of copper on the oxidase system has also been studied, but the concentration of copper was 30 mmol / Even at high concentrations such as L, the effect has been found to be very small [eg , Clin. Chem. 28, pages 578-588 (1982)].   Surprisingly, according to the present invention, as described below, glycated proteins In the oxidase / peroxidase method for measuring Interference effect can be eliminated by using copper at a concentration lower than 0.1 mmol / L . Furthermore, in this system, when water is used as a sample other than serum or plasma, Copper directly oxidizes the chromogenic system. Therefore, the oxidation-reduction potential of copper is Must be higher than the reduction potential. Copper in the analysis of serum or plasma samples The reason for not interfering is that the product of proteinase digestion of blood proteins It is thought to be caused by   An object of the present invention is to produce peroxidase in the same reagent as proteinase. It is an object of the present invention to provide an enzymatic method for measuring glycated proteins in a physical sample. Amazing In particular, the activity of peroxidase is not affected by the proteinase. Since the other components of the second reagent do not significantly affect the absorbance of the cuvette, No correction using the sample is required. Absorbance change contains a certain amount of glycated protein What is necessary is just to compare with the change in absorbance when a calibration solution (calibrant) is used.   Another object of the present invention is to provide ascorbate or The purpose is to prevent interference with glycated protein measurement caused by bilirubin. The interference effect of ascorbate is that peroxidase is not the second reagent but the first It can also be sufficiently removed by adding it to a reagent.   The present invention relates to a process that requires peroxidase, Is generally applicable to various processes where it is advantageous to mix . Removing analytes from proteins and measuring with oxidase; or The present invention is also applicable when intact proteins interfere. The method of the present invention is biological It is also suitable for measuring a specific saccharified component in a liquid or glycated hemoglobin.   The present invention comprises the following steps (i) to (iii): Provides a quality measure:   (i) mixing a first reagent containing proteinase and peroxidase with a sample This produces a substrate that allows oxidation by ketoamine oxidase. ,   (ii) adding a second reagent containing ketoamine oxidase, and then   (iii) By measuring the hydrogen peroxide produced or the oxygen consumed, the sugar Detection and / or quantification of the immobilized protein.   The present invention relates to (i) a first reagent containing proteinase and peroxidase, (i) i) a second reagent containing ketoamine oxidase and (iii) an optional formed water peroxide For measuring glycated protein, comprising means for measuring oxygen or oxygen consumption Kits are also provided.   In general, the methods of the present invention involve biological fluids containing biological fluids, such as serum or plasma. Applicable to samples.   According to the present invention, the proteinase is generally proteinase K, preferably Proteinase K from Tritirachium album Peroxidase is generally horseradish peroxidase. Good More preferably, ketoamine oxidase is a Klebsiella bacterium, Fusarium spp. Or from fungi of the genus Acremonium or yeasts of the genus Debaryomyces Particularly preferred are those obtained from fungi of the genus Fusarium. (Ketoamineoxy The enzyme oxidizes the carbon atom at the 1-position of the sugar moiety of the glycated protein, thereby forming an amine bond. Of sugar ozone and hydrogen peroxide from amino acids by catalyzing hydrolytic cleavage of union . )   In general, the required measurements are optionally modified by the Trinder reaction (also called the “PAP” method). Or the use of oxygen electrodes.   In a preferred embodiment of the invention, the interference by ascorbate is present in the first reagent. Copper (II) compounds (preferably copper (II) acetate) and optional cholic acid and / or It can be prevented by incorporating sophenanthroline disulfonic acid, And / or bilirubin interfere with the first and / or second reagent. Inhibited by incorporating a Russian salt (preferably potassium ferrocyanide) can do. Further, from the viewpoint of maintaining the activity of ketoamine oxidase, the second Ethylenediaminetetraacetic acid and / or mannitol may be added to the reagent.   In a preferred embodiment of the present invention, a protein derived from Tritylachium album is used. The concentration of Nase K used in the cuvette is 1 to 10 g / L. The working concentration of luoxidase in the cuvette is 0.01-1 g / L.   The present invention is further described by the following examples.   Example 1   Two reagent pairs for glycated protein measurement were prepared. One reagent pair is in the first reagent Contains a peroxidase and a proteinase, and the other reagent pair contains Contains oxidase. In the case of the first reagent pair, the first reagent is (N-2-H (Droxyethyl) piperazine-N '-(3-propanesulfonic acid) (EPPS) 4-Aminoantigen dissolved in a buffer (pH 8.5) containing 0 mmol / L Pyrene (3.0 mmol / L), proteinase K (12 g / L) and horseradish The second reagent contained peroxidase (0.4 g / L) and 100 mmol / L of EPP. TOOS (26.6 mmol / L) and ketoamily dissolved in S buffer (pH 8.5) Oxidase (10000 U / L). In the case of the second reagent pair, the first The reagent was 4-aminoamine dissolved in a 100 mmol / L EPPS buffer (pH 8.5). Contains N. tipiprene (3.0 mmol / L) and proteinase K (12 g / L). The two reagents were prepared from TOOS (2) dissolved in 100 mmol / L EPPS buffer (pH 8.5). 6.6 mmol / L) and horseradish peroxidase (1.33 g / L) and Ketoamine oxidase (10000 U / L)It contains.   These reagents were tested using an S-type automatic analyzer manufactured by Cobas Mira. Protection 100 μL of the inase-containing reagent was placed in a plastic cuvette in a diabetic patient Was mixed with 10 μL of human serum and 40 μL of dilution water, and the inside of the sample tube was It was washed. After incubation at 37 ° C. for 7 minutes, 30 μL of the second reagent and 20 μL of dilution water was added into the cuvette to mix the contents. 550 of Cuvet The absorbance at nm was measured for 1.5 minutes at 25 second intervals after addition of the second reagent . The automatic analyzer automatically calculates the absorbance measurement result in consideration of the dilution when the second reagent is added. Correct dynamically.   Peroxidase is protected from proteinase by addition to second reagent FIG. 1 shows the results obtained by the formulation. Caused by glycated proteins in serum samples To calculate the change in absorbance due to the color of the peroxidase added. The change in absorbance of the water sample must be subtracted first.   Peroxidase as a component of the first reagent. FIG. 2 shows the results. The water sample does not show an increase in absorbance even with the addition of the second reagent. won. Despite the presence of proteinases and peroxidase, serum samples The absorbance change observed in the case was similar to the result in FIG. 1 with the water blank subtracted. .   Example 2   4-Aminoantigen dissolved in 100 mmol / L EPPS buffer (pH 8.5) Perene (2.25 mmol / L) and horseradish peroxidase (0.2 g / L) ), And a reagent containing or not containing proteinase K (12 g / L). Prepared. These reagents were stored at room temperature. Peroxidase in each reagent The activity is measured by the ability of the reagent to produce purprogalin from pyrogallol and hydrogen peroxide. The measurements were taken 0.5 and 24 hours after preparation. The results are shown below.   Proteinase K can be used for horseradish competition even when the reagent solution is stored at room temperature for 24 hours. It did not reduce oxidase activity.   Example 3   A two-part reagent for glycated protein measurement was prepared and stored at 4 ° C. The first reagent is 75 Sodium tartrate (1.2 mmol) dissolved in mmol / L EPPS buffer (pH 8.0) l / L), copper acetate (300 μmol / L), potassium ferrocyanide (100 μmol / L) , TOOS (8 mmol / L), peroxidase (0.4 g / L) and proteiner Contains ZeK (12 g / L). The second reagent was a 83 mmol / L EPPS buffer (pH 8). . 4-aminoantipyrene (10 mmol / L) and ketoamine Contains oxidase (10000 U / L).   These reagents were tested before storage at 4 ° C and after storage for 22 days. The test was performed using a sample. The test was performed using the automatic analyzer manufactured by Cobas Mira used in Example 1. I did it. Serum samples are stored frozen in aliquots and thawed immediately prior to analysis Aliquots were used for each analysis. Absorption due to glycated proteins in the sample The change in degree is measured by measuring the absorbance immediately before the addition of the second reagent 2.1 minutes after the addition of the reagent. Calculated by subtracting from the measured absorbance. The results are shown below.                                Absorbance change   Therefore, if the oxidase / peroxidase assay is completely performed For example, even if the reagent is stored at 4 ° C. for 22 days, the horseradish competition using proteinase K No signal decrease due to the degradation of luoxidase was observed.   Example 4   Peroxida for its ability to eliminate interference by ascorbate and bilirubin Three types of two-part reagents for glycated protein measurement were prepared to demonstrate the effect of lyse . In reagent system A, the first reagent was a 75 mmol / L EPPS buffer (pH 8.0). TOOS (8 mmol / L), peroxidase (0.4 g / L) and The second reagent contained Rotinase K (12 g / L), and 83 mmol / L of EPPS buffer. 4-aminoantipyrine (10 mmol / L) dissolved in a liquid (pH 8.0) and keto Containing amine oxidase (10000 U / L) (this reagent system Is not included). Reagent system B contains potassium ferrocyanide (100 μmol / L), copper acetate (300 μmol / L) and sodium tartrate (1.2 mmol / L) ) Is the same as the reagent system A except that) is added. These additives are bilirubin and asbestos It is a component that reduces the interference effect of corbate. Reagent system C is used as the first reagent. No peroxidase was added to the second reagent Same as reagent system B except for 1.33 g / L. Therefore, in the case of the first embodiment, After mixing the sample, reagent and diluent in the analyzer in this way, The concentration of peroxidase in the three reagent systems is the same in the three reagent systems.   The above three reagent systems are used to assay glycated proteins in the following four samples: Used: (1) water, (2) control serum dilution (1 part water for 4 parts serum), (3) ascord Serum diluent (ascorbin) diluted in the same manner using a stock solution of borate (Concentration of acid salt: 400 μmol / L), (4) Dilute using a stock solution of uncomplexed bilirubin. The diluted serum dilution (bilirubin concentration: 400 μmol / L). Obtained using reagent system C In calculating the results obtained, the change in absorbance obtained with the serum sample was Thus, by subtracting the obtained absorbance change, the peroxidase in the second reagent is obtained. Correction was made for absorbance due to zeolites.   In the table below, the interference effects by ascorbate and bilirubin are: The interference, expressed as a percentage of the absorbance change obtained by the control serum, It is shown as the resulting absorbance change.                          Percent recovery in serum   When used together with reagent system A, bilirubin (400 μmol / L) was used as a control sample. % To reduce the change in absorbance. However, reagent system B (peroxidized as the first reagent) In reagent system C (containing peroxidase in the second reagent) and in reagent system C (containing peroxidase in the second reagent). The interference effect is achieved by incorporating ferrocyanide, copper and tartrate into the first reagent. Is almost gone.   The interference effect by ascorbate is particularly large in reagent system A, 90% or more disappears. Due to the additional components of reagent system B, this effect is less than 5%. Is reduced. However, a trial in which an equal amount of peroxidase was added to the second reagent In the case of drug system C, the change in absorbance increases significantly. Therefore, in this system, Elimination of the interference effect by ascorbate is achieved by adding peroxidase to the first reagent. It depends on the addition.   Example 5   Tests should be performed as quickly as possible using an automated analyzer to maximize experimental efficiency. Should be done in However, some analysts believe that the first longer than three minutes The chemical reaction of the two-part reagent cannot be performed within the incubation time. Trial By reducing the time for incubating the sample with the first reagent. Detrimental effects on the ability to remove the interference effect of borate using reagent system A This will be described below. However, reagent system B significantly improves the ability to eliminate interference effects Contains additional components.   In reagent system A, the first reagent was a 75 mmol / L EPPS buffer (pH 8.0) Sodium tartrate (1.0 mmol / L), copper acetate (250 μmol / L), Potassium ferrocyanide (20 μmol / L), TOOS (8 mmol / L), peroxy Contains Dase (0.4 g / L) and Proteinase K (6 g / L). The second reagent is 4-Aminoantipyrene dissolved in 83 mmol / L EPPS buffer (pH 8.0) (10 mmol / L) and ketoamine oxidase (10000 U / L) . This reagent system uses different incubation times (2.9 minutes and 7 minutes) Tested.   In reagent system B, the first reagent was prepared in 75 mmol / LEPPS buffer (pH 8.0). Dissolved bathophenanthroline disulfonic acid (175 μmol / L), polyoxy Ethylene 10 tridecyl ether (1% w / v), cholic acid (2% w / v), copper acetate (1 00 μmol / L), potassium ferrocyanide (100 μmol / L), TOOS (8 mmol / L), peroxidase (0.4 g / L) and proteinase K (6 g / L). L). The second reagent has the same composition as in reagent system A.   Glycated proteins in the following three samples were assayed using the two reagent systems. : (1) control serum diluent (1 volume of water for 4 volumes of serum), (2) ascorbic acid Serum diluent (ascorbate) diluted in the same manner using a salt stock solution (Concentration: 300 μmol / L) and (3) diluted with a stock solution of uncomplexed bilirubin A diluted serum solution (concentration of bilirubin: 300 μmol / L).   Mix sample, reagent and diluent in the analyzer as in Example 1. (However, in this case, incubation of the sample and the first reagent was performed for 2.9 minutes. Or 7 minutes).   In the table below, the effects of ascorbate and bilirubin These interferences expressed as a percentage of the absorbance change obtained by the serum It is shown as the change in absorbance obtained with the agent.   Example 6   In the case of using a reagent for removing the above-mentioned interference effect, the sample and the ink of the first reagent The disadvantage of shortening the incubation time is that water was used as the sample. The reaction proceeds. This background reaction occurs when serum is the sample Although it is not clear, the above method is preferably performed using samples other than serum. No.   In reagent system A, the first reagent was a 75 mmol / L EPPS buffer (pH 8.0). Acetate (5 mmol / L) dissolved in water, bathophenanthroline disulfonic acid (144 μmol / L), polyoxyethylene 10 tridecyl ether (1.2% w / v) , Cholic acid (1.8% w / v), copper acetate (90 μmol / L), potassium ferrocyanide ( 90 μmol / L), TOOS (5.6 mmol / L), peroxidase (0.28 g / L) ) And proteinase K (4 g / L). The second reagent is 50 mmol / L EP 4-Aminoantipyrene (10.5 mmol / L) dissolved in PS buffer (pH 8.0) ) And ketoamine oxidase (13000 U / L).   Reagent system B except that disodium EDTA (30 mmol / L) was added to the second reagent Is the same reagent system as reagent system A.   Glycated proteins in the following samples were assayed using two reagent systems: (1) water (2) serum collected from diabetic patients and (3) plasma collected from diabetic patients.   The reagent system was tested using an S-type automatic analyzer manufactured by Cobas Mira. Proteiner 20 μL of a reagent containing 250 μL of the enzyme in a plastic cuvette And 30 μL of dilution water to wash the inside of the sample tube. 37 After incubation at 2.9 minutes at 50 ° C, 50 μL of the second reagent and 10 μL of dilution water were added at the same time. Mix in the cuvette. The absorbance of the cuvette at 550 nm is determined by adding the second reagent. The measurement was performed for 1.5 minutes at intervals of 25 seconds from the time.   The absorbance profiles obtained using reagent systems A and B are shown in FIGS. Shown respectively. When water is the sample, the increase in absorbance obtained after addition of the second reagent is: If EDTA is present in the second reagent, it will be blocked. Serum and plasma samples The signal obtained is 15% greater than when EDTA is present in the second reagent. No. All of these effects are thought to be due to the chelation of copper by EDTA. It is. Copper produces a signal in the presence of a chromogen and also produces ketoamine oxidase. Partially inhibits activity.   Example 7   The second reagent can be stabilized by the addition of mannitol. Two kinds Second reagents A and B were prepared. The second reagent A was an EPPS buffer (50 mmol / L; p H8.0), aminoantipyrene (10.5 mmol / L), EDTA (30 mmol / L) And ketoamine oxidase (6000 U / L). The second reagent B is man It has the same composition as the second reagent A except that it contains 5% of nititol. These reagents are frozen After drying and dissolving with demineralized water, all were stored at 25 ° C for 21 days, and then used as a control. And frozen at -20 ° C.   The stability of the mannitol-containing reagent and the mannitol-free reagent was determined according to Example 6. A serum sample and the following components containing Tested using one reagent: dissolved in 60 mmol / L EPPS buffer (pH 8.0) Calcium acetate (5 mmol / L), bathophenanthroline disulfonic acid (144 μm ol / L), polyoxyethylene 10 tridecyl ether (1.2%), cholic acid (1 . 8%), copper acetate (90 μmol / L), potassium ferrocyanide (90 μmol / L), TOOS (5.6 mmol / L), peroxidase (0.28 g / L) and protein Nase K (3 g / L). The results were measured 2.5 minutes after addition of the second reagent. From the absorbance at 0 nm, the absorbance of the cuvette at 550 nm immediately before the addition of the second reagent Every time Was calculated by subtracting   The change in absorbance after 21 days is as follows.   After storage at 25 ° C. for 3 weeks, the signal obtained when using reagent A was It dropped to 77% of the binding reagent. However, reagent B containing mannitol Was stable.   Example 8   Enzymatic assay for glycated proteins and nitro blue tetrazos in practical use Comparison of the lium method [Roche Cat. No. 0736694] with the furosine standard method Tested by: This method involves acid hydrolysis and subsequent Includes HPLC quantification of furosine specific for protein saccharification. full Cuctosyl lysine was used as a reference.   The composition of the enzyme reagent is as follows. The first reagent was a 60 mmol / L EPPS buffer. Calcium acetate (5 mmol / L) dissolved in a buffer solution (pH 8.0), bathophenanthro Phosphorus disulfonic acid (144 μmmol / L), polyoxyethylene-10-tridecyl Ether (0.25%), cholic acid (1.8%), copper acetate (30 μmol / L), ferro Potassium cyanide (90 μmol / L), TOOS (5.6 mmol / L), peroxida (0.28 g / L) and Proteinase K (4 g / L). The second reagent is Mannitol (3%) dissolved in 50 mmol / L EPPS buffer (pH 8.0), Ketoamine oxidase (9000 U / L), EDTA (30 mmol / L) and amino Contains noantivirene (10.5 mmol / L).   250 μL of the first reagent was placed in a plastic cuvette and 20 μL of the sample and The inside of the sample tube was washed by mixing with 30 μL of dilution water. 5 at 37 ° C After a 2 minute incubation, perform a second test in the same cuvette. The drug was mixed with 50 μL and 10 μL of dilution water. Absorbance of cuvette at 550 nm The degree was measured at 25 second intervals over 10 minutes. Due to glycated proteins in the sample The change in the absorbance of the second reagent was determined from the absorbance measured 5 minutes after the addition of the second reagent. It was calculated by subtracting the absorbance measured just before addition.   Glycated proteins contained in 56 serum samples collected from diabetic patients Assayed by method. In the enzymatic method and the NBT method, γ = 0.95 and And γ = 0.96, showing a good correlation with the standard method (see FIG. 5). 95μ for NBT method mol / L or a positive bias of 34% of the estimated upper limit of the standard. In that case, the regression line passes very close to the origin. This indicates that: You. That is, both the enzyme method and the NBT method measure the same analyte, but the former method uses serum Is not affected by the non-specific background reducing activity present in

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, I S, JP, KE, KG, KP, KR, KZ, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, S D, SE, SG, SI, SK, TJ, TM, TR, TT , UA, UG, US, UZ, VN (72) Inventor Polar, Sarah Catherine             UK, M15.8 El Davru             ー, Kent, Maidstone, Bear step             No. 176, Merton Road

Claims (1)

[Claims]   1. Glycated protein in a sample, which comprises the following steps (i) to (iii): Measurement method:   (i) mixing a first reagent containing proteinase and peroxidase with a sample This produces a substrate that allows oxidation by ketoamine oxidase. ,   (ii) adding a second reagent containing ketoamine oxidase, and then   (iii) By measuring the hydrogen peroxide produced or the oxygen consumed, the sugar Detection and / or quantification of the immobilized protein.   2. 2. The method according to claim 1, wherein the sample contains a body fluid, preferably serum or plasma.   3. The proteinase is proteinase K, preferably Tritylachium albu; The method according to claim 1, wherein the protein is proteinase K derived from a protein.   4. 2. The method according to claim 1, wherein the peroxidase is horseradish peroxidase. 3. The method according to any of 3.   5. If ketoamine oxidase is a Klebsiella bacterium, Fusarium or Cremonium fungus or Debaryomyces yeast (preferably Fusarium A ketoamine oxidase obtained from a fungus of the genus Genus. The method described in Crab.   6. Use of a modified Trinder reaction or the use of an oxygen electrode in the measurement process The method according to any one of claims 1 to 5, comprising:   7. Ascorbate interference is detected by using a copper (II) compound (preferably copper acetate (II )) And optional cholic acid and / or bathophenanthroline disulfonic acid The method according to any one of claims 1 to 6, wherein counting is performed by combining.   8. The bilirubin interference is added to the first reagent and / or the second reagent with a ferrocyanide salt ( (Preferably potassium ferrocyanide) Item 8. The method according to any one of Items 1 to 7.   9. In order to maintain the activity of ketoamine oxidase, ethylenediamine was added to the second reagent. The method according to any one of claims 1 to 8, further comprising mintetraacetic acid and / or mannitol. The method described.   10. (i) a first reagent containing proteinase and peroxidase, (ii) keto A second reagent containing an amine oxidase and (iii) an optional formed hydrogen peroxide or Characterized in that it has a means for measuring oxygen consumption, and a kit for measuring a glycated protein. .